As a HDL receptor, SR-BI is a key regulator in enhancing reverse cholesterol transport in the liver, and hepatic overexpression of SR-BI can decrease plasma levels of HDL cholesterol, which may have anti-atherosclerosis effects. One of the mechanisms by which FXR is involved in regulating cholesterol and bile acid homeostasis is via transcriptional regulation of target gene expression. FXR has previously been shown to induce SR-BI expression. However, the underlying molecular mechanism by which FXR induces SR-BI expression is not fully defined. Therefore, the purpose of the current study is to determine the molecular mechanism by which FXR regulates SR-BI expression in human and mouse models. The present study showed that SR-BI mRNA was induced in a FXR-dependent manner in mouse livers, primary human hepatocytes and HepG2 cells. Our results suggest that FXR regulates Sr-bi gene expression by binding to multiple IR1s in the first intron of the Sr-bi gene. Moreover, the serum HDL level was increased in FXR-KO mice when fed either a control or HFD. Increased Sr-bi mRNA levels were shown to be FXR-dependent under HFD feeding by direct binding of FXR to the first intron of the Sr-bi gene, which indicates that FXR may enhance HDL uptake into the liver via inducing Sr-bi gene transcription. Activation of FXR has been demonstrated to regulate the expression of many hepatic genes involved in lipid homeostasis, including SR-BI. Consistent with our findings, Sr-bi mRNA levels have been shown to increase in livers of C57BL/6J mice but not in livers of FXR-KO mice upon LCA and CA feeding. However, a conflict result has been reported that Sr-bi expression was L-Ascorbyl 6-palmitate reduced following administration of CDCA or GW404764 both in vivo and in vitro. The authors further demonstrated that the decrease of Sr-bi was mediated by the FXR/RXR-SHP-liver receptor homologue 1 pathway. These conflicting results may be due to the different experimental models and/or ligands used. The underlying molecular mechanisms of FXR in regulating SR-BI and the role of SR-BI in FXR-mediated lipid homeostasis are still not clear. Recently, a vast database of nuclear receptor binding sites has been established with the development of genome-wide discovery of Terutroban transcription factor binding sites by ChIP-on-chip and ChIP-seq techniques. These data have shown that transcription factors tend to bind to multiple sites in the promoter and/or enhancer regions of target genes. Although our original ChIP-seq data did not show FXR binding to the promoter region of Sr-bi, we found three FXR binding sites within the first intron of Sr-bi gene. Moreover, even though luciferase activity did not correlate with the abundance of FXR binding, all three of these novel response elements, which include IR1s, were demonstrated functional.

These results indicate that activation of FXR could induce Sr-bi transcription by directly binding to multiple IR1s located in Sr-bi gene. However, a recent study showed that FXR up-regulated Sr-bi in mouse hepatocytes through a FXR-pJNK-hepatocyte nuclear factor 4 a SR-BI pathway, which indicates FXR may regulate SR-BI in both direct and indirect manners. In addition to FXR, another nuclear receptor, peroxisome proliferator-activated receptor a, can also increase Sr-bi Anemarsaponin-BIII expression in liver of rats. PPARa has also been shown to be activated by FXR in HepG2 cells, which may represent another indirect mechanism of FXR in induction of Sr-bi expression in human. However, activation of PPARa in mouse livers by fibrates decreased hepatic Sr-bi protein expression without changing Sr-bi mRNA levels. The posttranscriptional regulatory effect of fibrates on murine hepatic Sr-bi protein levels was further demonstrated PPARa dependent using PPARa deficient mice. These controversial results on Sr-bi regulation by activation of PPARa may due to species-specific differences. Even though no IR1 was found in the human SR-BI gene compared to the mouse gene, our results still showed that activating FXR increased SR-BI expression in primary human hepatocytes and a human hepatoma cell line. One recent study demonstrated that FXR directly activates SR-BI gene transcription by binding to a DR8 motif in the promoter region of the human SR-BI gene, which may help in understanding the underlying molecular mechanisms of FXR in regulating human SR-BI expression. Increasing studies also have shown that a variety of nuclear receptors, including liver X receptors, LRH-1, peroxisome proliferator-activated receptor c, and HNF4a can stimulate hepatic SR-BI gene expression in humans. Thus, FXR may also modulate SR-BI expression through regulating or interacting with these nuclear receptors or signaling pathways. These data suggest that activation of FXR could up-regulate hepatic SR-BI transcription either directly or through coordinating the activity of other nuclear receptors in both mouse and human livers. Hepatic SR-BI has been shown to serve as a key mediator of RCT by taking of HDL cholesterol to the liver. A series of studies using transgenic or recombinant adenovirus-mediated mice showed that hepatic over-expression of SR-BI markedly reduces atherosclerosis. Furthermore, SR-BI KO mice have higher HDL cholesterol in the circulation and enhanced atherosclerosis Hexamethonium Bromide development. These results suggest that hepatic SR-BI is critical in protecting against atherosclerosis development. Our studies showed that, compared to WT mice, FXR-KO mice had more serum total and HDL cholesterol.

Meanwhile, the RTCA assay could directly distinguish different strains of the same virus on the basis of our results. For quantitation of virulence, it is necessary to compare different viral stains, determine the number of infectious units required to produce the specific endpoint. Taken together, it is indicated that the RTCA system may provide a feasible in vitro format for assessment of virulence by monitoring CPE kinetics in future investigations of the influenza virus. It may further help to elucidate the relationship between genetic alterations and virulent variations for virological surveillance of influenza. Since virus-induced CPE could be quantitatively monitored using the RTCA system, we also performed a real-time neutralization assay for measuring H1N1-specific antibodies in human sera using the RTCA system. Since the HI test is still used as a standard for epidemiological and immunological studies, as well as for measuring the efficacy of influenza vaccines and potency of neutralizing antibodies, this new assay was evaluated against the standard HI test on a panel of sera collected from adult donors before and after immunization with the H1N1 vaccine. Both assays showed an obvious increase in neutralizing antibodies in the test sera at 3 weeks post-vaccination, and there was a good agreement between the HI and NT antibody titers. However, higher antibody titers were detected using the RTCA-based NT method, indicating the higher sensitivity of this assay. This may have been due to the difference between the CPE-based and HAbased assays, which had been observed in previous reports. Such functional quantitation may provide a valuable platform for serological diagnosis, immunological study, or evaluation of vaccines for influenza. An HI titer of 1:40 is commonly recognized as representing protective immunity. Our results revealed 30% of the HI titers in the pre-vaccination sera were equal to 1:40, and two were even higher. The first H1N1 case was identified in May 2009 in Shanghai, and population-wide vaccination followed in October 2009. Hence, widespread exposure to H1N1 in the society may have caused the higher antibody titers in the donors detected here. In addition, other reports have suggested that serum cross-reactive antibody responses to H1N1 occurred after vaccination with seasonal influenza vaccine and that this also existed in the population during the pre-pandemic period. In many settings influenza is recognized as a major cause of disease and death worldwide, which is the first infectious disease with global surveillance. Not the 2009 Influenza A Virus but other concomitant seasonal or highly pathogenic avian influenza viruses have posed considerable threads to public health. Global surveillance and annual vaccination are both of the key strategies and measures for the prevention and control of influenza.

However, in recent years more emphasis has been given to the study of invertebrate endocrine system showing that many steroid metabolic pathways are common to the ones of vertebrates and that some of the sex steroids have conserved functions in invertebrates reproduction as well. Further studies are required but our results might indicate a possible mechanism of endocrine Epimedin-B disruption in E. albidus, where steroid and retinol metabolisms were disturbed, producing Bullatine-B imbalanced levels of reproduction hormones. From the uniquely affected transcripts after atrazine exposure, a gene coding for a histone was significantly up-regulated at the EC10. This protein is involved in biological processes related with cell adhesion or the regulation of cell shape and maintenance of DNA integrity. The functions of histones have also been linked to positive regulation of growth rate and larval development, and its enhancement was reported in a study where Caenorhabditis elegans was exposed to atrazine. Some studies with atrazine have also reported disruption on the mitochondrial electron flow. Owen et al. observed significant up-regulation of several transcripts coding for the oxidative phosphorylation pathway in Lumbricus rubellus. The proteomic approach used by Thornton et al. in Drosophila melanogaster exposed organisms also showed significant changes on the mitochondrial protein expressions. In our microarray results, transcripts coding for proteins of the electron transport system were mainly up-regulated after the EC20, confirming the assumption that atrazine affects the normal mitochondrial functioning. Along with carbendazim, atrazine also seems to affect the carbohydrate metabolism by enhancing gluconeogenesis. Both glucan endo-1,3-beta-glucosidase and larval visceral protein d transcripts were up-regulated after carbendazim and atrazine exposures, with validated expression levels by qPCR for the first mentioned transcript. This tendency for increased glucose storage has also been described in a study by Zaya et al. where gene expression coding for glycolysis in Xenopus laevis suggested inhibition of this energetic process. Carbendazim was the only pesticide to induce transcripts encoding for intermediate filament proteins which are involved in DNA ligation during DNA repair. Those transcripts are significantly up-regulated at all carbendazim concentrations suggesting DNA damage and the indication of potential genotoxic effect of this pesticide, even at low concentrations. Effects of carbendazim on reproduction have been attributed to its well known function to interfere with the assembly of microtubules, rather than a mechanism involving endocrine disruption. Our results seem to be in good agreement with that hypothesis. Stathmin 1 oncoprotein 18 and several tubulin transcripts, that are differentially expressed by this compound, code for proteins directly involved in the regulation of cellular proliferation by assembling/disassembling microtubules. Stathmin 1 gene encodes for a cytoplasmic tubulin-binding phosphoprotein that acts to sequester tubulin and favour microtubule disassembly. Disturbances in the normal expression of stathmin correlate with a decreased inactivation of tubulin, a constant microtubule and mitotic spindle assembly and a consequent incapacity to regulate cell cycle progression. For this reason, disturbances in stathmin 1 expression have been associated with several types of cancer. In the present study, the microtubule assembly/disassembly process seems to be affected not only by carbendazim but also by dimethoate.

Many of the upregulated inflammatory genes detected here have been shown to play an important role in the immune response to mycobacterial infection and several of these direct the transition from innate to adaptive Nimorazole immunity during infection. Indeed, the GO categories identified via systems analysis of the differentially expressed genes here suggest that the expression of macrophage genes involved in the communication between innate and adaptive immune cell types is a key biological process that occurs within the first 24 hours of M. bovis challenge in vitro. For example, TNF-a is a key pleiotropic cytokine produced by macrophages that plays an important role in granuloma formation and maintenance by inducing IFN-c release from T cells, which in turn, activates anti-mycobacterial function in infected macrophages. IL12 encodes a proinflammatory cytokine produced by macrophages that activates NK cells and T cells to produce IFN-c and promote the adaptive immune response to mycobacterial infection. IL-1b is a proinflammatory cytokine secreted largely by innate immune cells in response to mycobacterial infection and studies have shown that IL-1b is produced in excess at the site of infection, suggesting that it plays an important role in granuloma formation and maintenance. Genes encoding b-chemokines participate in early immuno-protection against mycobacterial pathogens following infection. IL-6, a pleiotropic inflammatory cytokine expressed by a wide range of immune cells including macrophages in response to mycobacterial infection, has been proposed to play an important role in protection against tuberculosis. IL6 displayed large fold-upregulation at all three time points analysed in the M. bovischallenged MDM, while the gene encoding the IL-6 receptor displayed Clopidol downregulation expression in the M. bovischallenged MDM at 2 hours and 24 hours and was not differentially expressed at the 6 hour time point. This suggests that endogenous IL-6 production by macrophages acts in an endocrine manner, presumably by initiating immune responses in other innate or adaptive immune cells. Indeed, IL-6 has been proposed as a key regulator of the immunological switch between innate and adaptive immune processes. Despite the role of chemokines and cytokines in directing the host immune response to control mycobacterial infection, there is evidence to suggest that their function can be used by mycobacterial pathogens to enable persistence within the host. For example, non-regulated production of TNF-a in lung tissue can result in immunopathology, including destructive inflammation and necrosis, allowing dissemination of the pathogen from infected cells. Furthermore, studies have shown that IL-6 can inhibit T cell responses following infection of macrophages with M. bovis-BCG and M. avium subspecies paratuberculosis, while other investigations have reported that M. tuberculosisinduced IL-6 production inhibits the anti-microbial activity of macrophages in response to IFN-c. IL10, which encodes an anti-inflammatory cytokine that limits local cytokine-induced tissue damage and systemic inflammatory responses during infection, was upregulated at 2 hours and 24 hours postchallenge. Interestingly, upregulation of IL10 resulting in the subsequent suppression of host innate immune responses to infection has been proposed as a mechanism which enables enhanced mycobacterial intracellular proliferation.